Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), affects more than one million people in North America. Increasing evidence suggests that an imbalance of Th1 / Th2 polarization, in favor of Th1 cell subsets, may be a key pathogenic mechanism in a variety of organ-specific autoimmune disease, such as IBD. In CD, evidence has accumulated from both animal models and human studies to indicate that Th1 cytokines are involved in the pathogenesis of this disorder. However, identification of the specific initiating factor(s) driving Th1-mediated immune responses represents an important issue. The present proposal focuses on IL-18, a newly described cytokine, primarily produced by macrophages and other non-immune cell types. Recent studies suggest that IL-18 may function as a "classic" proinflammatory cytokine by playing a primary role in Th1-mediated immune responses and strongly implicates IL-18 as a possible mediator of organ-specific autoimmune disease(s), including CD. Therefore, the central hypothesis of this proposal is that IL-18, having the ability to elicit Th1-polarized T cell responses, may play a crucial pathogenic role in CD, as well-defined prototypic Th1 disorder. The following four specific aims will be investigated to: 1) Characterize the expression of IL-18 in the intestinal mucosa of IBD patients. Using various techniques and at multiple levels, IL-18 mRNA and protein levels will be determined in tissues isolated from the intestines of IBD patients as well as inflammatory and normal controls (IC and NC, respectively). The results obtained from these studies will be compared to those from freshly isolated intestinal mucosal cells, including intestinal epithelial cells (IEC), lamina propria mononuclear cells (LPMC) and various mesenchymal cell populations. Finally, IL-18 expression will be correlated with disease severity as well as clinical phenotype of IBD subgroups. These studies, although descriptive, represent a fundamental step before pursuing more mechanistic studies described in the subsequent specific aims. 2) Determine the factor(s) regulating IL-18 production in, as well as the effects of IL-18 on, intestinal mucosal cells. The regulation of IL-18, in general, and in the context of intestinal inflammation, in particular, will be assessed by investigating the effects of several "classic" proinflammatory and Th1 cytokines on IL-18 expression in different gut mucosal cells. Furthermore, the effects of IL-18, itself, will be evaluated for its ability to activate the inflammatory response typical of CD in IEC, LPMC and intestinal mesenchymal cells by determining IL-18-induced transcription factor activation and subsequent cytokine profile expression. These experiments will reveal critical functions of IL-18 in regulating gut immune responses. 3) Determine the transcriptional regulation of the human IL-18 gene in different intestinal mucosal cell populations. Isolation of the human IL-18 gene, characterization of its structure and mapping of its transcriptional start sites(s) will be initially achieved in order to perform the analyses of IL-18 promoter activity. Promoter function studies will achieved the creation of reporter constructs, site mutagenesis experiments and transfection assays in different intestinal cells lines. These studies will define how the human IL-18 gene is regulated, particularly during gut inflammation, and if transcriptional control varies among different intestinal cell types. 4) Identify, and characterize the properties of, the 31 kD putative IL-18 homologue predominantly found in intestinal epithelial cells. In order to identify the true nature of this putative IL-18 related protein, an expression cDNA phage library will be derived from IEC and immunoscreened for IL-18+ phage plaques. Purified isolates will be subsequently characterized for their unique features. These experiments will allow the discovery of novel IL-18 homologues and determine if they are differentially expressed in various cell types within the intestinal mucosa. The ultimate goal of the present research proposal is to define the precise role of IL-18 in CD in order to develop specific treatment modalities aimed at modifying the natural course of this devastating disease.